Polycyclic ether antibiotic

ABSTRACT

A new polycyclic ether antibiotic with anticoccidial, insecticidal and antimicrobial activity, its production by fermentation and methods for its recovery and purification are described.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 548,421 filedFeb. 10, 1975, now U.S. Pat. No. 4,002,885.

BACKGROUND OF THE INVENTION

This invention is concerned with a new member of the acidic polycyclicether group of antibiotics, a class of compounds characterizedbiologically by their effect on cation transport in mitochondria. Thisfamily of antibiotics includes monensin (J. Amer. Chem. Soc., 89:5737,1957); nigericin (Biochem. Biophys, Res. Comm., 33:29, 1968); grisorixin(J. Chem. Soc. Chem. Commun., 1421, 1970); dianerycin (J. Antibiotics,22:161, 1969); salinomycin (J. Antibiotics, 27:814, 1974); X-537A (J.Chem. Soc. Chem. Commun., 967, 1972); X-206 (J. Chem. Soc. Chem.Commun., 927, 1971); and A204A (J. Amer. Chem. Soc., 95:3399, 1973).

The polycyclic ether antiboiotics listed above are active againstGram-positive bacteria, fungi and protoza. These antibiotics exhibitpotent anticoccidial activity.

The control of coccidiosis continues to be a serious problem to thepoultry industry. There are six species of coccidia which produce easilydiscernible morbidity in susceptible chickens. Eimeria tenella, E.necatrix. E. brunetti, E. acervulina, E. maxima and E. mivati producedamage either directly through destruction of epithelial cells of thedigestive tract or indirectly through production of toxins. Three otherspecies of protoza belonging to the same genus are considered to berelatively innocuous; however, E. mitis, E. hagani and E. praecox arecapable of reducing weight gain, lowering feed efficiency and adverselyaffecting egg production.

The polycyclic ether antibiotics possess a high degree of effectivenessagainst all species of Eimeria. These antibiotics can, therefore, beregarded as "broad spectrum" coccidiostats.

SUMMARY OF THE INVENTION

This invention is concerned with a new acidic polycyclic etherantibiotic produced by the submerged aerobic propagation in aqueousnutrient media of Streptomycea flaveolus ATCC 31100 Isolated from a soilsample from Japan. The antibiotic and its cationic salts are activeagainst a variety of microorganisms and are effective in controllingcoccidiosis in poultry. In addition to the above effects, thisantibiotic and its metallic salts also possess insecticidal activity.

DETAILED DESCRIPTION OF THE INVENTION

The antibiotic producing microorganism of the present invention wasfound on examination to have the morphological features of aStreptomyces. It was, therefore, planted on media designated for theInternational Streptomyces Project descriptive work by Shirling andGottlieb in International Journal Systematic Bacteriology, 16:313-340,1966 and also on a number of other media frequently used inidentification and compared with Streptomyces flaveolus ATCC 3319.

Incubation was at 28° C. except where noted otherwise, and results wererecorded at appropriate times; results given here are after two weeks ofincubation except where otherwise noted.

The media and reference to their composition are listed as follows:

1. Czapek-Sucrose Agar: Waksman, The Actinomycetes, Vol. II, 1961,medium no. 1, p. 328.

2. Glucose-Asparagine Agar: Waksman, The Actinomycetes, Vol. II, 1961,medium no. 2, p. 328.

3. Calcium Malate Agar: Waksman, Bact. Reviews, 21:1-29, 1957.

4. Nutrient Agar: Waksman, The Actinomycetes, Vol. II, 1961, medium no.14, p. 330.

5. Yeast Extract- Malt Extract Agar: Pridham et al., Antibiotics Annual,1956/1957: 947-953.

6. Gelatin: Gordon and Mihm, J. Bact., 73:15-27, 1957.

7. Starch Agar:

    ______________________________________                                        Potato Starch              20.0   g                                           NH.sub.4 Cl                0.5    g                                           Distilled Water            1      liter                                       Agar                       15     g                                                           pH 7.0                                                        ______________________________________                                    

8. Oatmeal Agar: 30 g Quaker Oats steamed in 1000 ml distilled water for30 minutes, strained, volume restored to 1 liter, pH adjusted to 6.5-7.0and 15 g agar added.

9. Tryptone Yeast Extract Broth: Pridham and Gottlieb, J. Bact.,56:107-114, 1948.

10. Cellulose: Jensen, Proc. Linnean Soc. N. S. Wales, 55:231-248, 1930,and medium no. 2511 in Levine and Schoenlein's A Compilation of CultureMedia for the Cultivation of Microorganisms, 1930.

11. Dextrose Nitrate Broth: Waksman, The Actinomycetes, Vol. II, 1961,medium no. 1 on p. 328 without agar.

12. Organic Nitrate Broth: Gordon and Mihm, J. Bact., 73:15-27, 1957.

13. Skimmed Milk: Difco.

14. Peptone Iron Agar: Difco.

15. Potato Plugs: Plugs placed in tubes containing a glass ring at thebottom and about 0.5 ml water and autoclaved at 121° C. for 20 minutes.

16. Carbon Utilization: Pridham and Gottlieb, J. Bact., 56:107-114,1948.

The new culture (Pfizer F.D. 24306) was described as follows on thevarious media (color with all of names capitalized are those of Ridgway,Color Standards And Nomenclature, 1912):

    __________________________________________________________________________                   Aerial            Soluble                                      Medium Growth  Mycellum  Reverse Pigment                                      __________________________________________________________________________    Gelatin                                                                              Good, flat                                                                            Cream colored with                                                                      Light yellow                                                                          Lacking                                                     gray developing in                                                            the center.                                                    Glucose-                                                                             Poor to Lacking   Creamy yellow                                                                         Lacking                                      Asperagine                                                                           moderate, flat,   color                                                Agar   creamy yellow                                                                 color, soft                                                            Czapek-                                                                              Good, slightly                                                                        White but becoming                                                                      Creamy yellow                                                                         Pale yellow                                  Sucrose                                                                              raised; earthy                                                                        pale gray around                                               Agar   odor    edge of colony                                                 Nutrient                                                                             Moderate, flat                                                                        White     Yellow  Lacking                                      Agar                                                                          Yeast extract-                                                                       Good, raised;                                                                         Fluffy to cottony,                                                                      Yellowish                                                                             Pale brown                                   malt extract                                                                         earthy odor                                                                           near Light Olive                                                                        brown                                                Agar           Gray                                                           Oatmeal Agar                                                                         Excellent,                                                                            Lacking except at                                                                       Whitish Lacking                                             flat; faint                                                                           edges of colony                                                                         gray                                                        fruity odor                                                                           where it was slight-                                                          ly raised and near                                                            Pale Mouse Gray.                                               Calcium                                                                              Moderate,                                                                             Slightly cottony                                                                        Whitish gray                                                                          Lacking                                      Malate slightly                                                                              surface, near Olive                                            Agar   raised; faint                                                                         Gray                                                                  fruity odor;                                                                  malate digested                                                        __________________________________________________________________________

Skimmed Milk-No. coagulation in 21 days; complete hydrolysis between14th and 21st days, pH changed from 6.5-6.7 to 7.1 after 21 days;pinkish tan soluble pigmeat.

Potato Plugs -- Growth moderate with slight roughening and white bloomon surface; gray soluble pigment.

Spores -- Oatmeal agar plates revealed chales of spores scattered alongthe bypass as loops, hooks and spirals of one to several moderatelytight turns, 10-50, spores per chain. Spores were broadly elliptical tonearly round, 1.1 × 1.1 to 2.7 × 1.6 but mostly 1.6 × 1.6 g. roughenedas if with short spires (compound microscope, 1000 ×); with scanningelectron microscope projections on the surface of the space were likethose shown in FIG. 38 is Int. Jr. System, Esct. 15: 60-159, 1965 ofcarbon replica of hairy spores of Streptomyces flavcolus. Spores atfirst were separated by connections narrower than the spores, butcontinued incubation for several weeks resulted in disappearance ofconnective cells.

Biochemical Properties -- No retaining production; H₂ 5 produced inthree days; gelatin liquified, starch hydrolysis only as a clear areaunder the colony; nitrate reduced to nitrite in dextrose nitrite brothbut not in organic nitrate broth; good growth on cellulose strips but nodisintegration; utilization of glucose, 1.(-) arabiaose, dextrin, D(-)fructose, D(+) galactose, glycerol, isositol, isulin, lactose, maltose,D(-) michitol, raffinose, thacaose, salicin, D(-) sorbitol, starch,sucrose, trehalose, D(-)xylose; dulcitol not uilized; utilization ofsorbose doubtful.

The new culture and the type strain Streptomyces fluaveolus ATCC 3319were essentially alike on plates of media for morphological study,showed no color change in the soluble pigment when the pH was changed,showed the same type of spore ornamentation, the same carbon utilizationpattern and nearly the same biochemical properties.

The greatest difference between the two cultures were reduction ofnitrate to nitrite in organic nitrate broth and the production of abright yellow soluble pigment on many media by Streptomyces flaveolusATCC 3319; minor differences in color or form of growth of the twocultures were noted.

The new culture (Pfizer F.D. 24306) was submitted to the American TypeCulture Collection in Rockville, Md. on Nov. 15, 1974 and given thedisignation Streptomyces flaveolus ATCC 31100. The permanency of thedeposit and ready accessibility thereto by the public are afforded inthe event the patent is granted. Access to the culture is availableduring pendency of the application under Rule 14 and 35 USC 112. Allrestrictions on the availability to the public of the culture depositedwill be irrevocably removed upon granting of the patent.

Cultivation of Streptomyces flaveolus ATCC 31100 preferably takes placein aqueous nutrient media at a temperature of 28-36° C., and undersubmerged aerobic conditions with agitation. Nutrient media which areuseful for such purposes include a source of assimilable carbon such asaugars, starches and glycerol; a source of organic nitrogen such ascasein, enzymatic digest of casein, soybean meal, cotton seed meal,peanut meal, wheat gluten, soy flour, meat meal and fish meal. A sourceof growth substances such as grain solubles and yeast extract as well assalts such as sodium chloride and calcium carbonate and trace elementssuch as iron, magnesium, zinc, cobalt and manganese may also be utilizedwith advantageous results. If escessive foaming is encountered duringfermentation, antifoam agents such as vegetable oils or silicones may beadded to the fermentation medium. Aeration of the medium in tanks forsubmerged growth is preferably maintained at the rate of about 1/2 to 2volumes of free air per volume of broth per minute. Agitation may bemaintained by means of agitators generally familiar to those in thefermentation industry. Aseptic conditions must, of course, be maintainedthrough the transfer of the organism and throughout its growth.

Inoculum for the preparation of the antibiotic may be obtained byemploying growth from a slant of the culture. The growth may be used toinoculate either shake flasks or inoculum tanks or the inoculum tanksmay be seed from the shake flasks. Growth in shaken flasks willgenerally have reached its maximum in 3 to 5 days whereas inoculum insubmerged inoculum tanks will usually be at the most favorable period in2 to 3 days. Substantial antibiotic activity is obtained in the finalfermentor stage in approximately 3 to 5 days. The antibiotic levelsrange from 50 to 500 mg per liter.

The process of antibiotic production is conveniently followed duringfermentation by biological assay of the broth employing a sensitivestrain of Staphylococcus aureus or Bacillus subtilis. Standard plateassay technique is employed in which the zone of inhibition surroundinga filter paper disc saturated with the broth is used as a measure ofantibiotic potency.

Thin layer chromatography employing silica gel is a useful tool foranalyzing the antibiotic produced in fermentation media and thecomposition of crude and purified materials extracted from thefermentation broths. The thin layer chromatograms, after developmentwith ethyl acetate, are sprayed with 3% vanillin in ethanolic sulfuricacid (98:5:1.5% v/v) followed by heating at 60°-80° C. for a fewminutes. The antibiotic is observed initially as a dirty brown spotchanging with time to a brilliant purple spot on a white background.

Antibiotic Compound 38,986 may be separated and recovered fromfermentation broth by extracting with an organic solvent such aschloroform, ethyl acetate or methyl isobutyl ketone. The major portionof the antibiotic is contained in the mycelium and may be extractedtherefrom by slurrying the separated mycelium with a water-solublesolvent such as methanol.

The preferred method of separation and recovery of antibiotic Compound38,986 is as follows: The whole (unfiltered) fermentation broth is twiceextracted with about 1/5 to 1/2 volume of methyl isobutyl ketone. Thesolvent extract is concentrated under vacuum to an oily residue which isthen slurried with silica gel PF₂₅₄ in heptane and added to a silica gelcolumn (preferably a bed of silicon gel 60 topped with a layer of silicagel PF₂₅₄, both available from E. Merck, Darmstadt, Germany). The silicagel column is successively developed with heptane, benzene, chloroform,chloroform:ethyl acetate (1:1 v/v), ethyl acetate and methanol. The mainantibiotic fraction is eluted with chloroform: ethyl acetate (1:1 v/v).The eluate is concentrated under vacuum, taken up in acetone and stirredfor about 30-60 minutes with activated charcoal (Darco G 60). Thecharcoal is removed by filtration, and the solution is concentratedunder vacuum to a foam which could not be induced to crystallize.

The antibiotic that is isolated at this stage is a mixture of the freeacid and the sodium and potassium salts of Compound 38,986 formed withboth sodium and potassium ions occurring in and scavenged from thefermentation broth.

The free acid of Compound 38,986 may be derived from the mixedsodium/potassium salts by adjusting the pH of an aqueous acetonesolution of the salts with dilute phosphoric acid. The acetone is thenremoved in vacuo and the aqueous phase extracted with ethyl acetate. Theorganic phase after drying over sodium sulfate is evaporated in vacuo toa foam.

The sodium salt of Compound 38,986 may be obtained by adjusting anaqueous acetone solution of the free acid to pH 8.5 with sodiumhydroxide. The potassium salt is similarly obtained using potassiumhydroxide.

Compound 38,986 and its salts exhibit excellent activity againstcoccidiosis infections in poultry. When incorporated in the diet ofchickens at a level of 50 to 200 ppm, the compounds are effective incontrolling single infections of Kimeria tenella, E. acervulina, E.maxima, etc. and mixed infections of these organisms.

Because of its end use for the prevention andtreatment of coccidiosis inpoultry, whole fermentation broth containing Compound 38,986 may betaken to dryness (preferably by spray-drying) and incorporated inpoultry feed at the desired antibiotic potency level.

Compound 38,986 and its salts also exhibit excellent activity againstactoparasites. For example, the mortality of blowflies which come intocontact with a solution of Compound 38,986 in a concentration of 25 ppmis 100%.

Compound 38,986 exhibits inhibitory action against the growth of anumber of microorganisms (Table I). The test organism is inoculated in aseries of test tubes containing nutrient medium and variousconcentrations of Compound 38,986 to determine the minimal concentrationof the antibiotics in meg/ml which inhibits the growth of the organismover a period of 24 hours.

                                      Table I                                     __________________________________________________________________________                      Compound 38,986                                                                          Compound 38,986                                  Organism          (mixed Na/K salts)                                                                       (free acid)                                      __________________________________________________________________________    Trepodern hyodynenterine                                                                        0.39       --                                               Bacteroides fragilis 78A009                                                                     6.25       --                                               Bacteroides fragilis 78A014                                                                     6.25       --                                               Bacteroides fragilis 78A021                                                                     6.25       --                                               Bacteriodes fragilis 78A024                                                                     6.25       --                                               Staphylococcus aureus 01A005                                                                    0.39       0.78                                             01A052            0.39       0.39                                             01A110            0.39       0.39                                             01A111            <0.10      0.20                                             01A087            <0.10      0.78                                             01A400            0.39       0.39                                             Streptococcus faccalis 02A006                                                                   < 0.10     < 0.10                                           Streptococcus pyogenes 020203                                                                   < 0.10     < 0.10                                           Mycobacterium smegratis 05A001                                                                  3.12       3.12                                             Bacillus subtilis 06A001                                                                        < 0.10     < 0.10                                           Escherichia coli 51A229                                                                         > 200      > 200                                            Pseudomenas aeruginosa 52A104                                                                   > 200      > 200                                            Klebsiella pneumoniae 53A009                                                                    > 200      > 200                                            Proteus mirabilis 57C064                                                                        > 200      > 200                                            Salmonella cholerae-suis 58B242                                                                 > 200      > 200                                            Pasteurella multocida 59A001                                                                    25         25                                               Serratia marceseens 63A017                                                                      > 200      > 200                                            Enterobacterium acrogenes 67A040                                                                > 200      --                                               Neisseriae sicca 66C000                                                                         <0.10      < 0.10                                           __________________________________________________________________________

Efficacy data for compound 38,986 and its salts against coccidiosisinfection in chickens are obtained in the following manner. Groups of3-5 ten day old SPF white leghorn cockerel chicks are fed a mash dietcontaining Compound 38,986 or one of its salts uniformly dispersedtherein. After being on this ration for about 24 hours, each chick isinoculated per os with oocysts of the particular species of Eimeriabeing tested. Other groups of chicks fed on an antibiotic-free mash dietare similarly infected and serve as infected controls. Non-infected,non-medicated chicks serve as normal controls. The results of treatmentare evaluated after 5 days in the case of E. acervulina and 6 days forall other Elmeria species.

Table II illustrates the results obtained with the mixed sodium andpotassium salts of Compound 38,986.

                  Table II                                                        ______________________________________                                        Species                Ave. degree                                            infection    Dose (ppm)                                                                              of infection*                                                                            Ratio*                                      ______________________________________                                        Eimeria tenella                                                                            200       1.30       0.37                                                     150       2.70       0.77                                                     100       1.30       0.37                                                     75        2.70       0.77                                                     50        3.00       0.86                                        Eimeria accryaliae                                                                         200       1.00       0.50                                                     150       1.40       0.70                                                     100       2.00       1.00                                                     75        1.20       0.60                                                     50        1.40       0.70                                        Eimeria necatrix                                                                           200       0.60       0.30                                                     350       1.80       0.90                                                     100       0.60       0.30                                                     75        1.20       0.60                                                     50        1.40       0.70                                        Eimeria maxima                                                                             200       1.60       1.00                                                     150       0.80       0.50                                                     100       0.60       0.38                                                     75        1.20       0.75                                                     50        1.60       1.00                                        Eimeria brunetti                                                                           200       0.60       0.27                                                     150       1.00       0.45                                                     100       1.00       0.45                                                     75        0.60       0.27                                                     50        1.40       0.64                                        Mixed Infection                                                                            200        0.40, 0.0**                                                                             0.20, 0.0                                   (Coccivac D, Sterwine                                                                      150       0.80, 0.80 0.40, 2.0                                   Laboratories, Opelika,                                                                     100       0.80, 0.80 0.40, 2.0                                   Alabama)     75        1.40, 1.20  0.70, 3.00                                              50        1.40, 0.40  0.70, 1.00                                 ______________________________________                                         *The criteria used to measure anticoccidial activity consisted of lesion      scores of 0 to 4 for E. tenella after J.E. Lynch (A new method for the        primary evaluation of anticoccidial activity. Am. J. Vet. Res.,               22:324-326, 1961); and 0 to 3 for the other species based on a                modification of the scoring system devised by J. Johnson and W. H. Reid       (Anticoccidial drugs. Lesion scoring techniques in battery and floor pan      experiments in chicks. Exp. Parasit., 28:30-36, 1970). A constant ratio       was established by dividing the lesion score of each treated group by the     lesion score of the infected control.                                         **The first number pertains to intestinal lesions and the second number t     cecal lesions.                                                           

Similar coccidial control may be obtained by incorporating the free acidof Compound 38,986, the sodium salt, or the potassium salt thereof inthe poultry diet. An efficient and economical method of treatmentcomprises the incorporation of dried fermentation medium containingantibiotic Compound 38,986 in the poultry diet at the desired antibioticpotency level.

The insecticidal activity of Compound 58,986 is illustrated by thefollowing tests against a number of insects.

Twenty females of the World Health Organization standardfully-susceptible strain of Musca domestica, 2-4 days old, areanaesthetized with carbon dioxide. One microliter of a solutioncontaining Compound 38,986 is applied to the dorsal surface of thethorax. The flies are then maintained in gauze-covered pots at 25° C.and approximately 50% relative humidity for 24 hours with a cotton woolpad moistened with sugar solution and placed on the gauze as food.Twenty flies are treated with solvent only and serve as controls. At theend of this period the mortality is noted and recorded as a percentageafter correction for any mortality among the controls.

Five grams of sugar are covered with acetone and treated with 0.5 ml ofa solution containing 12.5 to 100 parts per million of Compound 38,986.The acetone is then evaporated off with constant stirring. The sugarbait is then transferred to a plastic pot covered with gauze, and twentyadult female Lucilia sericata are introduced. A pad of wet cotton woolprovides a free water supply and the insects are maintained for 24 hoursand mortalities calculated. The control group comprises twenty fliesmaintained with untreated sugar.

0.5 ml of Compound 38,986 test solution is pipetted evenly on to Whatmanno. 1 filter paper (8 cm. × 6.25 cm.) to give a deposit of 50 to 100mg/m². When dry, the paper is rolled and slid into a test tube to whichis added 1.5 ml of calf serum which is absorbed by the filter paper andserves as food. Fifteen Lucilia sericata larvae, approximately 36 hoursold, are introduced and the tube closed with a cotton wool plug andstored with the top part only in a strong light to keep the larvae inthe lower part of the tube and thus in contact with the filter paper.The insects are maintained for 24 hours, and mortalities calculated.Fifteen larvae confined with untreated filter paper serve as controls.

The test results are tabulated in Table III.

                  Table III                                                       ______________________________________                                        Musea domestica                                                                            Lucilia sericata                                                                            Lucilia sericata                                   (adult)      (adult)       (larvae)                                           Dose             Dose            Dose                                         (μ)/ferale                                                                          % Kill  (ppm)    % Kill (mg/m.sup.2)                                                                         % Kill                                ______________________________________                                        1        100     100      100    100    100                                   0.5      100     50       100     50     18                                   0.25     100     25       100                                                 0.125     40     12.5      40                                                 ______________________________________                                    

The above test results may be obtained with the sodium salt or thepotassium salt of Compound 38,986; a mixture of the free acid, thesodium salt and the potassium salt of Compound 38,986; or driedfermentation medium containing Compound 38,986 or its cationic salts.

EXAMPLE I

A sterile aqueous medium having the following composition was prepared:

    ______________________________________                                                             Grams/liter                                              ______________________________________                                        Glucose                    10.0                                               Soluble starch             20.0                                               Yeast extract              5.0                                                Enzymatic digest of casein 5.0                                                K.sub.2 HPO.sub.4          0.4                                                CaCO.sub.3                 4.0                                                                  pH - 7.0                                                    ______________________________________                                    

Cells from a slant of Streptomyces flaveolus ATCC 31100 were transferredto a series of 300 ml flasks each containing 50 ml of this sterilemedium and shaken on a rotary shaker for 3-4 days at 28°-30° C. Five mlaliquots of this grown inoculum were transferred aseptically to 300 mlflasks containing 100 ml of sterile medium as described above. Aftershaking for 3-4 days at 28°-30° C., the grown inoculum was transferredto four-liter fermentors containing two liters of the following sterilemedium:

    ______________________________________                                                             Grams/liter                                              ______________________________________                                        Glucose                    10.0                                               Starch                     10.0                                               Soy flour                  10.0                                               Grain solubles             5.0                                                NaCl                       5.0                                                CaCO.sub.3                 1.0                                                                pH - 7.0                                                      ______________________________________                                    

The fermentation was conducted for 90-120 hours at 28°-36° C. withstirring at 1700 revolutions per minute and aeration at about one volumeof air per volume of broth per minute. Large fermentors containing from8,000 to 10,000 gallons of medium may be inoculated with about 2% ofthis growth. The fermentation is conducted until an antibiotic potencyof at least 50 mg per liter is obtained (90 to 120 hours).

One hundred liters of whole broth were treated with one-fifth volume ofmethyl isobutyl ketone and the organic phase separated with the aid of acentrifugal separator. The organic layer derived in this fashion wasconcentrated in vacuo to an oily residue, 1.5 kilos of which weredispersed on silica gel by its addition in solution in 3 liters ofheptane to silica gel PF₂₅₄, 1.5 kilograms, and silica gel 60,250 grams.The resultant slurry was added to a bed of 500 grams of silica gel 60.The silica gel was then washed successively with 2 gallons of heptane, 2gallons of benzene, 1 gallon of chloroform, 2 gallons ofchloroform:ethyl acetate (1:1, v/v), 2 gallons of ethyl acetate and 1gallon of methanol. The desired antibiotic, Compound 38,986, was foundto reside in the chloroform:ethyl acetate (1:1, v/v) wash (93 grams).This was dissolved in 100 ml of heptane and added to a column of silicagel 60 (3 kilos) made up in heptane and the column developed withchloroform containing increasing proportions of ethyl acetate. Theappropriate fractions were combined, evaporated in vacuo (19 grams),taken up in acetone, 250 ml, and treated with an equal weight ofactivated charcoal (Darco G 60) at room temperature for about 60minutes. Filtration, followed by evaporation in vacuo yielded a foam (18grams) which could not be persuaded to crystallize. This material was amixture of the free acid, sodium salt and potassium salt of Compound38,986.

The physical and chemical data for the material isolated at this stagevaries from fermentation to fermentation because of the differingpercentages of free acid, sodium salt and potassium salt. The data fortwo representative samples are shown below:

Sample A is characterized by an average composition by weight of 64.05%carbon and 9.36% hydrogen, an optical rotation of [α] _(D) ²⁵° = - 7.3°(c = 1, acetone), and when pelleted in KBr, FIG. 1, exhibitingdistinguishable bands in the infrared spectrum over the region 2 to 14microns at the following wavelengths in microns: 2.98, 3.42, 5.75, 6.12,6.85, 7.25, 8.65, 9.00, 9.24, 9.40, 9.65, 10.10, 10.40 and 11.17.

Sample B is characterized by an average composition by weight of 66.96%carbon and 9.45% hydrogen, an optical rotation of [α] _(D) ²⁵° = - 2.1°(c = 1, acetone), and when pelleted in KBr, FIG. 2, exhibitingdistinguishable bands in the infrared spectrum over the region 2 to 14microns at the following wavelengths in microns: 2.95, 3.40, 5.72, 5.78,6.35, 6.82, 7.22, 7.57, 7.96, 8.28, 8.65, 8.88, 8.97, 9.22, 9.37, 9.65,9.82, 10.10, 10.38, 10.82, 11.15, 11.85, 13.37 and 13.72.

Sample A and Sample B are soluble in methanol, ethanol, acetone,chloroform methylene chloride, diethyl ether, ethyl acetate, and methylisobutyl ketone; partially soluble in heptane; and insoluble in water.

Both samples give the same free acid on treatment with dilute phosphoricacid.

EXAMPLE II

The mixture of the free acid and mixed sodium and potassium salts ofCompound 38,986 of Example I was dissolved in aqueous acetone and the pHadjusted to 4.5 with dilute phosphoric acid. The acetone was thenremoved in vacuo and the aqueous phase extracted with ethyl acetate. Theorganic phase was then dried over sodium sulfate and evaporated in vacuoto a foam. The free acid could not be induced to crystallize.

The free acid is soluble in methanol, ethanol, acetone, chloroform,methylene chloride, diethyl ether, ethyl acetate and methyl isobutylketone. It is partially soluble in heptane and insoluble in water.

The free acid has an optical rotation of [α]_(D) ²⁵° = 7.5° (c = 1,acetone), After drying overnight in vacuo at 70° C. over phosphoruspentoxide, the average composition by weight is 65.11% carbon and 9.54%hydrogen. The free acid of Compound 38,986 possesses no characteristicultraviolet light absorption pattern.

The infrared spectrum of the free acid of Compound 38,986, FIG. 3, isattached. A KBr pellet shows characteristic absorption in the infraredregion at the following wavelengths in microns: 2.98, 3.44, 5.75, 5.78,6.85, 7.24, 7.98, 8.30, 8.65, 9.00, 9.23, 9.42, 9.67, 9.83, 10.10,10.38, 10.83, 10.92, 11.18, 11.45, 11.85, 13.40, 13.70 and 14.10.

EXAMPLE III

The sodium salt of Compound 38,986 was obtained from the free acid ofExample II by adjusting an aqueous acetone solution of the free acid topH 8.5 by the addition of 1.0 N NaOH. The acetone was removed in vacuoand the aqueous phase extracted with ethyl acetate to yield a foam whichcould not be induced to crystallize.

The sodium salt of Compound 38,986 has an optical rotation of [α] _(D)²⁵° = - 4.9° (c = 1, acetone). After drying overnight in vacuo at 70° C.over phosphorus pentoxide, the average composition by weight is 64.15%carbon and 9.44% hydrogen.

The infrared spectrum of the sodium salt of Compound 38,986, FIG. 4, isattached. A KBr pellet shows characteristic absorption in the infraredregion at the following wavelengths in microns: 2.95, 3.42, 5.74, 5.78,6.35, 6.83, 7.23, 7.55, 7.98, 8.27, 8.67, 9.00, 9.25, 9.40, 9.66, 9.84,10.10, 10.37, 10.80 11.15, 11.45, 11.87, 13.42, 13.70 and 14.05.

EXAMPLE IV

The potassium salt of Compound 38,986 was prepared by the method ofExample III employing potassium hydroxide in place of sodium hydroxide.The material was obtained as a foam that could not be induced tocrystallize.

The optical rotation is [α] _(D) ²⁵° = + 8.3 (c = 1, acetone). Theaverage composition by weight of the dried sample is 63.26% carbon and8.91% hydrogen.

The infrared spectrum of the potassium salt of Compound 38,986, FIG. 5,is attached. A KBr pellet shows characteristic absorption in theinfrared region at the following wavelengths in microns: 3.00, 3.45,5.75, 5.80, 6.40, 6.85, 7.15, 7.27, 7.55, 7.79, 8.00, 8.15, 8.30, 8.74,9.00, 9.25, 9.67, 9.85, 10.13, 10.42, 10.82, 11.19, 11.40, 11.97, 12.30,12.90, 13.40 and 14.05.

EXAMPLE V

The method of Example I may be repeated employing clarified fermentationbroth in place of whole, unfiltered fermentation broth.

EXAMPLE VI

The mycelium separated from the clarified fermentation broth of ExampleV may be slurried several times with methanol, the methanol extractconcentrated under vacuum and the residue treated by the method ofExample I.

EXAMPLE VII

The fermentation process of the method of Example I may be repeatedemploying a fermentation medium of the following composition:

    ______________________________________                                                            Grams/liter                                               ______________________________________                                        Glucose                   20.0                                                Starch                    10.0                                                Soy flour                 10.0                                                Grain solubles            5.0                                                 Fe.sub.2 (SO4).sub.3      0.2                                                 MaCl.sub.2                0.2                                                 CaCO.sub.3                1.0                                                 NaCl                      5.0                                                 Methyl oleate             2.0                                                 Soybean oil               2.0                                                                pH - 6.6-6.7                                                   ______________________________________                                    

At the end of the fermentation cycle, the whole unfiltered fermentationbroth is taken to dryness, preferably by spray-drying.

What is claimed is:
 1. The method of controlling coccidiosis in poultrywhich comprises administering to poultry an effective amount forcontrolling coccidiosis of antibiotic Compound 38,986, or apharmaceutically acceptable cationic salt thereof incorporated in thediet of said poultry, said antibiotic compound when in the form as thefree acid having an optical rotation of α_(D) ²⁵° = - 7.5° at aconcentration of 1% in acetone; an average composition by weight of65.11% carbon, 9.54% hydrogen and 25.35% oxygen (by difference); and,when pelleted in KBr, exhibiting characteristic abosrption in theinfrared region at the following wavelengths in microns: 2.98, 3.44,5.75, 5.78, 6.85, 7.24, 7.98, 8.38, 8.65, 9.00, 9.23, 9.42, 9.67, 9.83,10.10, 10.38, 10.92, 11.18, 11.45, 11.85, 13.40, 13.70, and 14.10. 2.The method of claim 1 wherein said antibiotic compound is the sodiumsalt of Compound 38,986.
 3. The method of claim 1 wherein saidantibiotic compound is the potassium salt of Compound 38,986.
 4. Themethod of claim 1 wherein said antibiotic compound is a mixture of thefree acid, the sodium salt and the potassium salt of Compound 38,986. 5.The method of claim 1 wherein said antibiotic compound is Compound38,986.